Resistance to insulin is a feature of patients with type 2 diabetes mellitus (T2D) and the insulin resistance syndrome. PC-1, a class II plasma membrane exoprotein inhibits the IR alpha subunit in a region between residues 485-599. This IR region links the alpha subunit ligand binding domain to the beta subunit tyrosine kinase domain. In most subjects with insulin resistance, we and others have found PC-1 in muscle and other tissues is either over expressed or is in a more active form (Q allele). Transfection and overexpression of PC-1 into cultured cells selectively reduces both IR tyrosine kinase activity and IR signaling. We now find that human PC-1 overexpression in mouse muscle and liver causes in vivo insulin resistance and diabetes. We hypothesize, therefore, that PC-1 is a major cause of insulin resistance. Herein we plan to document that PC-1 is an important regulator of insulin action, define how PC-1 interacts with the IR, and employ strategies both in vitro and in vivo to antagonize PC-1. We propose the following: First, we plan to metabolically phenotypically characterize mice that are over expressing the various alleles of PC-1. We will employ mice with adenovirus-mediated PC-1 overexpression in liver, and transgenic mice with general and tissue-specific PC-1 overexpression. Second, employing our animal models of PC-1 overexpression, we will investigate whether anti PC-1 monoclonal antibodies, PC-1 RNAi, and PC-1 antisense oligomers will lower PC-1 levels and improve insulin action. To regulate PC-1 levels, we will also use the Tet off/on system. Third, because we have data both in vitro and in vivo indicating that PC-1 directly interacts with the IR alpha subunit, we will investigate the interactions of PC-1 with the IR by elucidating how and where PC-1 binds to the IR. For this purpose, we will employ direct binding studies. In addition, mutants of both the IR and PC-1 will be produced to locate discrete sites of protein-protein interaction. By defining the contact points between PC-1 and the IR, we have the potential to devise strategies to inhibit this interaction. Fourth, PC-1 content in cultured fibroblasts and muscle biopsy closely correlate. Therefore, employing fibroblasts from insulin resistant patients, the mechanisms that cause PC-1 overexpression in insulin resistant humans will be explored. We will determine therefore whether PC-1 overexpression in fibroblast is caused by transcriptional and/or post-transcriptional mechanisms.